1) Field of the Invention
The present invention relates to a method for assaying bilirubin fractions in living body fluid samples such as plasma, serum, urine, etc. and more particularly to a method for assaying bilirubin non-.delta. fractions and a bilirubin .delta. fraction in living body fluid samples, utilizing bilirubin oxidase incapable of oxidizing a bilirubin .delta. fraction but capable of oxidizing bilirubin non-.delta. fractions in the presence of at least one of a cationic surfactant and a nonionic surfactant, and also relates to an assay kit for use in the same method.
2) Description of the Related Art
Bilirubin is a metabolic product of hemoglobin originating from aged erythrocyte and constitutes the main component of bile pigment. Blood contains a fraction with increased water solubility (conjugate form) due to ester linkage of side chain propionate group of bilirubin mainly to glucuronic acid enzymatically in the liver and a fraction with decreased water solubility (free form) due to the propionate group remaining in a free state. The former is called "direct bilirubin" because of easy reaction with a diazo reagent, whereas the latter is determined as "indirect bilirubin" as subtracting the concentration of direct bilirubin from the concentration total bilirubin obtained by diazo dying of bilirubin in the presence of a reaction accelerator such as alcohol, etc., because reaction with the diazo reagent occurs only in the presence of the reaction accelerator.
By fractionally determining the concentrations of respective conjugate form and free form bilirubin, diacrisis and diagnosis of jaundice due to various liver diseases, hemolytic diseases, etc. can be made and thus assaying of bilirubin is an important item of examination in the clinical laboratory test.
Apart from the reactivity with a diazo reagent, fractional analysis of bilirubin by high performance liquid chromatography (HPLC) has been also studied. Bilirubin in serum is separated mainly into 4 fractions, i.e., .alpha., .beta., .gamma. and .delta. fractions by HPLC, where the .alpha. fraction is identified as free form bilirubin; the .beta. fraction as bilirubin, one of whose two propionate groups in the molecule undergoes ester linkage to glucuronic acid (bilirubin monoglucuronide); the .gamma. fraction as bilirubin, whose two propionate groups all undergo ester linkage to glucuronic acid (bilirubin diglucuronide); and the .delta. fraction as bilirubin in a covalent bond with albumin. Furthermore, it is presumed that the .delta. fraction results from non-enzymatic reaction of the .gamma. fraction with albumin (Toshio Yamashita: Journal of The Japanese Society of Internal Medicine 78 (11), 36-41 (1989)).
According to the classification based on reactivity with a diazo reagent, it is presumed that the .alpha. fraction corresponds to indirect bilirubin and the .beta. and .gamma. fractions and also the .delta. fraction correspond to direct bilirubin (John J. Lauff: Clin. Chem. 28 (4), 629-637 (1982)).
The .beta. and .gamma. fractions are constituted from water-soluble low molecular weight compounds and thus can readily pass through renal glomeruli to be rapidly discharged into urine. That is, they never remain in blood for a long time. The .delta. fraction, on the other hand, can undergo metabolism not as bilirubin but rather as albumin, and it is presumed that the half life of the .delta. fraction in blood is as long as 2-3 weeks, whereas that of other fractions is as short as a few ten minutes (John J. Lauff: J. Chromatogr., 226, 391-402 (1981); J. S. Weis: N. Engl. J. Med., 309, 147-156 (1983)).
Assay results of serum bilirubin according to the conventional diazo method can supply important information on the initial phase of jaundice, particularly hyper-(direct form)bilirubinemia, but the diazo method has been so far considered to be uncapable of giving its assay results reflecting correctly and timely the progress changes in jaundice. This phenomenon is obviously due to the fact that reaction of the .gamma. fraction with albumin can increase the .delta. fraction, apparently retarding the bilirubin metabolic rate, thereby causing a lag between the bilirubin assay results and the actual amelioration and/or deterioration of the disease. That is, it can be said that in case that there are a large amount of .delta. fraction having the slow metabolic process and also showing the reactivity with direct bilirubin in samples, the bilirubin assay results according to the diazo method have no more clinical significance, because the assay results involve also the high level .delta. bilirubin and will fail therefore to correctly reflect the progress of the disease. (Toshio Yamamoto: Tan-Kan-Sui (Gallblader, Liver and Pancrease), 11 (3), 393-400 (1985)).
In order to determine bilirubin concentrations sensitive to changing states of disease, attempts have been made for fractional quantitative determination of individual .alpha. to .delta. fractions, one of which is based on the above-mentioned HPLC method (John J. Lauff: J. Chromatogr., 226, 391-402 (1981)). Its improvements have been made by omitting complicated sample pretreatment steps as much as possible (Nakamura H.: Bunsekikagaku, 36, 352-355 (1987); Yukihiko Adachi: Gastroenterologia Japanica, 23 (3), 268-272 (1988); Yuko Kato: Medical Journal of Kinki University, 14 (1), 97-112 (1989)).
It was reported on the basis of these HPLC results that a ratio of individual bilirubin fractions such as .delta./(.beta.+.gamma.+.delta.), (.alpha.+.gamma.)/.delta. or .beta./.delta. sensitively reflects changing states of liver diseases and is particularly useful for prognostication. Assay of individual bilirubin fractions could solve the problems of bilirubin assay by the conventional diazo method, because information even on progress of state of disease could be obtained by only one assay, and thus has been regarded as having lifted the position of serum bilirubin in the liver function test (Toshio Yamamoto: Kan-Tan-Sui (Gallblader, Liver and Pancrease), 11 (3), 393-400 (1985)).
However, the HPLC method requires about one hour in the assay of one sample, even if the complicated pretreatments could be omitted, and thus can be regarded not as a practical method for assaying a large number of samples daily, but as a laboratory-scale research method.
On the other hand, Wu et al developed a dry chemistry method (Ektachem method) as a highly practical method for assaying bilirubin fractions on the basis of such a principle that proteins including the .delta. fraction can be separated and removed by a special membrane; then the low molecular weight bilirubin fraction having permeated through the membrane is made to react with cationic polymers; the free form bilirubin (.alpha. fraction) and the conjugate form bilirubin (.beta.+.gamma. fraction) can be fractionally and quantitatively determined at 400 nm and 460 nm, respectively, as a result of a change in the reflectance spectrum from each other, where the .delta. fraction can be obtained by subtracting the assay results of the free form bilirubin (.alpha. fraction) and the conjugate form bilirubin (.beta.+.gamma. fraction) from the assay result of total bilirubin obtained by the diazo method. That is, according to the Ektachem method, a total bilirubin concentration, a free form bilirubin (.alpha. fraction) concentration, a conjugate form bilirubin (.beta.+.gamma. fraction) concentration and a .delta. fraction concentration are fractionally and quantitatively determined, respectively (Tai-Wing Wu: Clin. Chem., 30 (8), 1304-1309 (1984)).
The Ektachem method can assay one sample in the order of a few minutes and thus can be regarded as a practically distinguished method for assaying fractions. It was reported that the assay results of the Ektachem method were in good agreement with those of the HPLC method in the hyper-(direct form)bilirubinemia, upon comparison thereof. However, it was also reported that in case that the total bilirubin concentration is in the normal range (not more than 1.3 mg/dl) or in case of hyper-(indirect form)bilirubinemia the assayed .delta. fraction of the Ektachem method considerably deviated from that of the HPLC method (Yukihiko Adachi: Gastroenterologia Japonica, 23 (3), 268-272 (1986)).
Furthermore, the Ektachem method has such a disadvantage as a strong influence of colored substances because of measurement of reflectance intensity (Young D. S.: Washington D.C. AACC Press (1990); Yound D. S.: 1991 Supplement. Washington D.C. AACC Press (1991); Friedoman R. B.: Washington D.C. AACC Press (1990)). Furthermore, special instruments are required for the assay due to the dry chemistry, and thus the Ektachem method lacks the versatility in simultaneous multiphasic assay of a large number of samples as in the multiphasic assay in the clinical laboratory test, as another disadvantage.
Another practical method for assaying bilirubin fractions was proposed by Akira Kosaka: Seibutsu Shiryo Bunseki (Biological Sample Analysis), 9 (3), 15-23 (1986); JP-A-62-105047) or by Umachi et al (Hisami Umachi: Rinsho Byori (Clinical Pathology), 37 (8), 905-910 (1989)).
According to the Kosaka et al method, bilirubin oxidase derived from Myrothecium verrucaria is made to act on a sample at pH 10.0 to eliminate the .beta. and .gamma. fractions by oxidation, and then only the .delta. fraction is subjected to diazo coloration by direct diazo reaction for assaying, or both .alpha. and .delta. fractions are subjected to diazo coloration by indirect diazo reaction (diazo reaction in the presence of a reaction accelerator such as alcohol, etc.) for assay and the assay result is subtracted from the assay result of total bilirubin concentration or the assay result of direct bilirubin concentration, whereby concentrations of the .alpha. fraction and the .beta.+.gamma. fraction as well as the .delta. fraction can be determined.
However, the Kosaka et al method suffers from such inconveniences that at the same time of eliminating the .beta. and .gamma. fractions by oxidation with the bilirubin oxidase derived from Myrothecium verrucaria, a portion of the .alpha. fraction is likewise eliminated by oxidation; a portion of the .alpha. fraction also undergoes diazo coloration during the direct diazo reaction; the degree of reaction differs between the direct reaction and the indirect reaction in the diazo coloration of the .delta. fraction, which result in very complicated errors in the assay results, showing a tendency of deviation of assayed .delta. fraction concentration from that of the HPLC method (Akira Kosaka: Seibutsu Shiryo Bunseki (Biological Sample Analysis), 9 (3), 15-23 (1986); John J. Lauff: Clin. Chem., 28 (4), 629-637 (1982); Lo H. D.: Clim., 29, 31-36 (1983)).
The Kosaka et al method is of multi-stage reaction process and basically manual assay is obligatory. Thus, the method is not applicable to an all-purpose, two-reagent automatic analyzer and lacks simplicity.
According to the Umachi et al method, on the other hand, bilirubin oxidase derived from Trachyderma tsunodae is made to act on a sample at pH 8.0 in the presence of salicylic acid to eliminate the .alpha., .beta. and .gamma. fractions by oxidation and only the .delta. fraction is subjected to diazo coloration by diazo reaction for assay. Assay of the individual fractions can be carried as in the Kosaka et al method, that is, concentrations of the .alpha. fraction and the .beta.+.gamma. fraction as well as the .delta. fraction can be determined from the assay result of .delta. fraction concentration or by subtraction from the assay result of total bilirubin fraction concentration or assay result of direct bilirubin fraction concentration.
In the Umachi et al method, bilirubin oxidase derived from Trachyderma tsunodae eliminates about 40% of the .delta. fraction in the presence of salicylic acid by oxidation and the exact determination of .delta. fraction is impossible to conduct. Furthermore, the method is also of multi-stage reaction process and basically manual assay is obligatory. Thus, the method is not applicable to an all-purpose automatic analyzer and lacks simplicity.
In the assay of serum bilirubin, as described above, the conventional diazo method cannot exactly reflect the state of disease in case of larger proportion of the .delta. fraction, but only assay results of bilirubin fractions can sensitively respond to the state of disease. Only technical break through to the assay of bilirubin fractions is obviously a development of simple method for assaying the .delta. fraction. However, in the daily clinical laboratory test, no suitable reagents for assaying the .delta. fraction have been available yet for an all-purpose two-reagent automatic analyzer capable of conducting simultaneous multi-item assay of a large number of samples. Thus, development of reagents capable of assaying the .delta. fraction with a high reliability for assay in the automatic analyzer has been keenly desired.